This invention is related to the field of pathogen-inducible promoters, and chimeric DNA sequences comprising said promoters, especially in the area of plant biotechnology.
Inducible promoters include any promoter capable of increasing the amount of gene product produced by a given gene, in response to exposure to an inducer. In the absence of an inducer the DNA sequence will not be transcribed. Typically, the factor that binds specifically to an inducible promoter to activate transcription is present in an inactive form which is then directly or indirectly converted to the active form by the inducer. The inducer may be a chemical agent such as a protein, metabolite (sugar, alcohol, etc.), a growth regulator, herbicide, or a phenolic compound or a physiological stress imposed directly by heat, salt, wounding, toxic elements etc., or indirectly through the action of a pathogen or disease agent such as a virus. A plant cell containing an inducible promoter may be exposed to an inducer by externally applying the inducer to the cell such as by spraying, watering, heating, or similar methods. Inducible promoters are known to those familiar with the art and several exist that are be used to drive expression of genes of interest. Examples of inducible promoters include the inducible 70 kD heat shock promoter of Drosophila melanogaster (Freeling, M. et al., Ann. Rev. Genet. 19, 297-323) and the alcohol dehydrogenase promoter which is induced by ethanol (Nagao, R. T. et al., in: Miflin, B. J. (ed.) Oxford Surveys of Plant Molecular and Cell Biology, Vol. 3., pp. 384-438, Oxford Univ. Press, 1986). Examples for promoters that are inducible by a simple chemical are the promoters described in WO 90/08826, WO 93/21334, WO 93/031294 and WO 96/37609.
An important subclass of inducible promoters are the promoters which are induced in plants upon pathogen infection. As examples of a pathogen-inducible promoter the PRP1 promoter (also named gst1 promoter) obtainable from potato (Martini N. et al. (1993), Mol. Gen. Genet. 263, 179-186), the Fis1 promoter (WO 96/34949), the Bet v 1 promoter (Swoboda, I., et al., Plant, Cell and Env. 18, 865-874, 1995), the Vst1 promoter (Fischer, R., Dissertation, Univ. of Hohenheim, 1994; Schubert, R., et al. Plant Mol. Biol. 34, 417-426, 1997), the sesquiterpene cyclase promoter (Yin, S., et al., Plant Physiol. 115, 437-451, 1997) and the gstA1 promoter (Mauch, F. and Dudler, R., Plant Physiol. 102, 1193-1201, 1993) may be mentioned. A drawback of some of these promoters is that they are also active constitutively or that they do not react to certain types of pathogens. Furthermore, it would be advantageous to have promoters that regulate expression very soon after pathogen infection, i.e. with as short as possible induction times.
Thus, there is still need for promoters that are pathogen-inducible which overcome the disadavantages of the prior art.
We now have found DNA fragments which are the upstream regulatory regions for plant genes coding for hexose oxidase, capable of promoting pathogen-inducible transcription of an associated DNA sequence when re-introduced into a plant. Preferably such a fragment is obtainable from Helianthus annuus. Said DNA fragment specifically is the upstream regulatory region of the gene coding for hexose oxidase, denoted as MS59, more specifically characterized in that it comprises the nucleotide sequence from 1 to 1889 depicted in SEQ ID NO: 15.
Also part of the invention is a DNA fragment obtainable from Lactuca sativa, capable of promoting pathogen-inducible transcription of an associated DNA sequence when re-introduced into a plant, specifically the DNA fragment that it is the upstream regulatory region of the gene coding for hexose oxidase, denoted as WL64 (SEQ ID NO: 18).
Also included in the invention is a portion or variant of a DNA fragment according to any described above, capable of promoting pathogen-inducible transcription of an associated DNA sequence when re-introduced into a plant.
Embodiments of the invention are chimeric DNA sequences comprising in the direction of transcription a DNA fragment according to any one of the DNA fragments described above and a DNA sequence to be expressed under the transcriptional control thereof and which is not naturally under transcriptional control of said DNA fragment. A preferred embodiment is such a chimeric DNA sequence wherein the DNA sequence to be expressed causes the production of an antipathogenic protein, which is preferably selected from the group consisting of chitinase, glucanase, osmotin, magainins, lectins, saccharide oxidase, oxalate oxidase, toxins from Bacillus thuringiensis, antifungal proteins isolated from Mirabilis jalapa, Amaranthus, Raphanus, Brassica, Sinapis, Arabidopsis, Dahlia, Cnicus, Lathyrus, Clitoria, Allium seeds, Aralia and Impatiens and albumin-type proteins, such as thionine, napin, barley trypsin inhibitor, cereal gliadin and wheat-alpha-amylase.
Another embodiment of the chimeric DNA sequences of the invention is a chimeric DNA sequence wherein the DNA sequence to be expressed causes the production of a protein that can induce a hypersensitive response, preferably selected from the group consisting of CF and Pto proteins from tomato, avr proteins from Cladosporium fulvum and elicitor proteins from Pseudomonas or Xanthomonas.
Further part of the invention are replicons comprising above mentioned chimeric DNA sequences preferably having at least one recognition site for a restriction endonuclease for insertion of a DNA sequence to be expressed under the control of said DNA fragment. Also included in the invention are microorganisms containing such a replicon, plant cells having incorporated into their genome a chimeric DNA sequence according to those described above, and plants essentially consisting of said cells. such a plant is preferably a dicotyledonous plant. Also part of said plants selected from seeds, flowers, tubers, roots, leaves, fruits, pollen and wood, form part of the invention.
Yet another embodiment of the invention is the use of a DNA fragment as described above for identifying homologues capable of promoting pathogen-induced transcription in a plant.
Further use of a chimeric DNA sequence according to the invention for transforming plants and use of a portion or variant of the DNA fragments according to the invention for making hybrid regulatory DNA sequences is part of the invention.
Another object of the invention is the use of a chimeric DNA sequence as described above for conferring pathogen resistance to a plant.